VPR: Vpr is a conserved multi-functional protein that is incorporated into virions and drives early (transport of pre-integration complex) and late (apoptosis) viral life cycle steps. Despite more than 20 years of research, the mechanism by which Vpr accelerates virus replication is not well understood [40-42]. A wide range of molecular functions has been ascribed to Vpr, including roles in G2 cell cycle arrest and pre-integration complexes [43-46], perhaps reflecting a multi-purpose adaptor protein. Recently, SAMHDI, a deoxynucleoside triphosphate triphosphohydrolase, was found to be a myeloid and dendritic cell restriction factor that is overcome by HlV-2 Vpx [47, 48], a parajog of Vpr believed to carry out some functions of Vpr. Like Vif and Vpu, both Vpr and Vpx interact with the host ubiquitin machinery. Vpr binds the Cullin4 RING Box Ubiqultin Ligase (CRL4) complex [49, 50], suggesting that Vpr may overcome additional host restrictions via the degradation pathway. Due to its central roles in a number of key HIV infection processes, Vpr represents an exciting target for structure-function studies and positions Vpr as a viable therapeutic target. With no universally accepted model for its functions, however, only by clearly defining the host proteins and pathway(s) perturbed by Vpr at the molecular level-as outlined in this proposal-will the bona fide targetable activity of Vpr be revealed. The HARC Proteomics/Genomics Core compiled the most extensive Vpr-host interaction data set described to date. Encouragingly, these novel interactions reveal host complexes and partners (e.g. dynein and C0P9) functionally linked with many Vpr activities. By evaluating the functional significance and ability to form structured complexes, we will develop and test new hypotheses about Vpr-host interactions. PR: Many viruses encode proteases that disarm host defenses or hijack host processes in addition to cleaving viral substrates. For example, poliovirus PR inhibits host protein synthesis by cleaving translation initiation factors, while rhinovirus PR cleaves the nuclear pore complex and adaptor proteins involved in the innate immune response [51-58]. HIV-1 PR processes the viral Gag and GagPol polyproteins during virion maturation, but its role in affecting host functions is unclear. In vitro, PR cleaves several mammalian proteins [59-65], but it is not known if cleavage occurs during HIV infection or how much active enzyme exists in host cells during the viral life cycle [66, 67]. The HARC Center Proteomics/Genomics Core uncovered novel host proteins that interact with, an inactive version of PR, including proteins involved in immune responses, splicing, translation, cell growth, and apoptosis [1, 29]. Active PR in cells cleaved some of these proteins, including a single site in the RNA binding domain of the d subunit of the eukaryotic translation initiation factor 3 complex (elF3d). elF3d was cleaved nearly as efficiently as Gag. An intriguing hypothesis is that elF3d recruits the translation initiation complex to the entering HIV genome, obstructing reverse transcription. Our structural studies of such PR-host complexes will be the first of PR with a macromolecular substrate, providing new knowledge on substrate recognition. Our analyses of the roles of potential host PR substrates in HIV replication have the potential to establish new paradigms for PR function, link PR to the roles of Vit Vpu, and Vpr, and define the structures of novel PR-host complexes.